PROJECT SUMMARY Almost 6 million Americans currently live with Alzheimer's disease (AD), and this number is projected to increase to 14 million by 2050. The consensus is arising in the field that in order to treat AD effectively, early detection at the MCI stage is a must. Thus, there is a critical need for a novel minimally invasive and cost-effective diagnostic assay capable of such early detection. Cell-secreted extracellular vesicles (EVs) recently gained significant attention in the fields of liquid biopsy and AD research. Within the scope of biomarker detection, EVs offer numerous benefits for clinical analysis, including non-invasive collection, a suitable sample source for longitudinal disease monitoring, ability to cross the blood-brain barrier, higher stability and sample volumes, faster processing times and lower cost. Multiple reports linked EVs to neurodegeneration and have been able to couple exosomal load in CSF to AD progression. However, the analysis of plasma exosomes and other EV has not really been possible for early AD detection. The procedures for plasma biomarker analysis are very long and cumbersome due to extremely low target levels and high background of free plasma proteins. In order to enable better biomarker discovery and AD diagnosis, a more reliable and efficient approach is needed, capable of enriching potential AD-associated proteins with higher purity. In this NIH SBIR Phase I study, we will develop a novel method for fast and reproducible capture and isolation of EVs with >95% recovery yield and >99.9% purity from plasma samples. We propose to implement this EV capture approach to detect and quantify the presence of common AD markers (amyloid-?, p-tau) in plasma EVs, and for discovery of new protein and phosphoprotein biomarkers in plasma EVs. We will also correlate and match their levels in CSF EVs and as soluble proteins to evaluate the clinical relevance of using plasma EVs as the source for diagnostics. The following aims will be completed in the Phase I of the proposal: Aim #1: Develop and validate the EVtrap method for plasma EV isolation and purification. Aim #2: Implement EVtrap for detection of common AD biomarkers in plasma and CSF exosomes. Aim #3: Develop EVtrap for discovery of plasma exosome proteins and phosphoproteins as new AD biomarkers. By the completion of this project, EVtrap capture beads method will be optimized, an AD biomarker discovery platform from plasma EVs will be developed, and a minimally invasive early Alzheimer's disease detection assay will be validated that can overcome the limitations of current approaches, and thus could have an enormous public health impact and market potential.